B-cell cultivation method

ABSTRACT

Herein is reported a method for co-cultivating one or more B-cells comprising the step of incubating the one or more B-cells with EL4-B5 cells, whereby the EL4-B5 cells have been obtained/are from a cultivation of EL4-B5 cells that has a cell density of from 600,000 cells/ml up to 1,500,000 cells/ml.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/144,240, filed on Sep. 27, 2018, which is acontinuation of International Application No. PCT/EP2017/057250, filedon Mar. 28, 2017, the entire contents of which are incorporated hereinby reference, and which claims priority to EP 16162954.8, filed on Mar.30, 2016.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically as a text file in ASCII format and is herein incorporatedby reference in its entirety. Said text file, created on Nov. 25, 2020is named “P33508-1_US_Seq_Listing.txt” and is 8,694 bytes in size.

FIELD OF THE INVENTION

Herein are reported methods for co-cultivating B-cells with EL4-B5feeder cells, which have been obtained from a culture of EL4-B5 cells ofa certain cell density, obtaining the amino acid sequence of at leastthe variable domains of a monoclonal antibody secreted by a singleB-cell, and for producing the antibody.

BACKGROUND OF THE INVENTION

For obtaining cells secreting monoclonal antibodies the hybridomatechnology developed by Koehler and Milstein is widely used. But in thehybridoma technology only a fraction of the B-cells obtained from animmunized experimental animal can be fused and propagated. The source ofthe B-cells is generally an organ of an immunized experimental animalsuch as the spleen.

Zubler et al. started in 1984 to develop a different approach forobtaining cells secreting monoclonal antibodies (see e.g. Eur. J.Immunol. 14 (1984) 357-363, J. Exp. Med. 160 (1984) 1170-1183). Thereinthe B-cells are obtained from the blood of the immunized experimentalanimal and co-cultivated with murine EL-4 B5 feeder cells in thepresence of a cytokine comprising feeder mix. With this methodology upto 50 ng/ml antibody can be obtained after 10-12 days of co-cultivation.

Weitkamp, J-H., et al., (J. Immunol. Meth. 275 (2003) 223-237) reportthe generation of recombinant human monoclonal antibodies to rotavirusfrom single antigen-specific B-cells selected with fluorescentvirus-like particles. A method of producing a plurality of isolatedantibodies to a plurality of cognate antigens is reported in US2006/0051348. In WO 2008/144763 and WO 2008/045140 antibodies to IL-6and uses thereof and a culture method for obtaining a clonal populationof antigen-specific B cells are reported, respectively. A culture methodfor obtaining a clonal population of antigen-specific B-cells isreported in US 2007/0269868. Masri et al. (Mol. Immunol. 44 (2007)2101-2106) report the cloning and expression in E. coli of a functionalFab fragment obtained from single human lymphocyte against anthraxtoxin. A method for preparing immunoglobulin libraries is reported in WO2007/031550.

In WO 2011/147903 a single B-cell cultivation method is reported.

In WO 2013/076139 CD40L expressing mammalian cells and their use arereported.

In WO 2013/092716 a rapid method for cloning and expression of cognateantibody variable region gene segments is reported.

In U.S. Pat. No. 7,807,415 methods for producing stable immortalizedB-lymphocytes are reported. In EP 0 488 470 methods for the productionof antibodies are reported.

Seeber, S., et al. reported a robust high throughput platform togenerate functional recombinant monoclonal antibodies using rabbitB-cells from peripheral blood (PLOS One 9 (2014) e86184/-14).

In US 2007/065919 methods of producing stable B-lymphocytes arereported.

In WO 2012/178150 methods for developing antigen-specificantibody-producing cell lines and monoclonal antibodies are reported.

Kwekkeboom, J., et al. reported an efficient procedure for thegeneration of human monoclonal antibodies based on activation of human Blymphocytes by a murine thymoma cell line (J. Immunol. Meth. 160 (1993)117-127).

Weber, M., et al,. reported combining EL4-B5-based B-cell stimulationand phage display technology for the successful isolation of humananti-Scl-70 autoantibody fragments (J. Immunol. Meth. 278 (2003)249-259).

Dohmen, S. E., et al., reported about the production of recombinant Igmolecules from antigen-selected single B cells and restricted usage ofIg-gene segments by anti-D antibodies (J. Immunol. Meth. 298 (2005)9-20).

SUMMARY OF THE INVENTION

Herein is reported a method for the co-cultivation of single depositedB-cells, which can be of any source, with feeder cells in a suitableco-cultivation medium.

The invention is based at least in part on the finding that the EL4-B5feeder cells used in the co-cultivation can be obtained from acultivation of EL4-B5 cells that has a cell density of more than 500,000cells/ml, especially in the range of 600,000 cells/ml up to 1,500,000cells/ml, i.e. the EL4-B5 cells have been cultivated up to said celldensity.

The invention is further based at least in part on the finding thatoverall the frequency of antigen binding and IgG positive wells amongstall IgG positive wells increases depending on the EL-4-B5 cultivationdensity at the time of harvest of the EL-4-B5 cells, i.e. if EL-4-B5cells obtained from EL4-B5 cultivations with higher cell densities atthe time of EL-4-B5 cell harvest are used in B-cell co-cultivations thebefore referred-to increase of frequency can be obtained. This effectwas observed when EL4-B5 cells obtained from a cultivation of EL4-B5cell with a cell density of up to 1,500,000 cells/ml were used in theco-cultivation. That is, using the same concentration of EL-4-B5 cellsas before in the co-cultivation results in an increased frequency ofantigen binding and IgG positive wells when the EL-4-B5 cells have beencultivated to final cell density of up to 1,500,000 cells/ml.

The individual aspects as reported herein are methods for

-   -   i) the isolation of a B-cell or a B-cell clone from a population        of B-cells, whereby the isolated B-cell or B-cell clone produces        an antibody specifically binding to a target,    -   ii) the co-cultivation of single deposited B-cells, and    -   iii) the production of an antibody.

Concomitantly with the methods also the corresponding uses are alsoencompassed and disclosed.

One aspect as reported herein is a method for co-cultivating one or moreB-cells comprising the step of

-   -   incubating the one or more B-cells with EL4-B5 cells, whereby        the EL4-B5 cells have been obtained/are from a cultivation of        EL4-B5 cells that has a (final) cell density of from 600,000        cells/ml up to 1,500,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a (final) celldensity of 650,000 cells /ml up to 1,450,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a (final) celldensity of 650,000 cells/ml up to 825,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a (final) celldensity of 1,400,000 cells/ml up to 1,500,000 cells/ml.

One aspect as reported herein is a method for co-cultivating one or moreB-cells comprising the steps of

-   -   cultivating EL4-B5 cells to a cell density of from about 600,000        cells/ml to about 1,500,000 cells/ml, and    -   incubating the one or more B-cells with an aliquot of the EL4-B5        cells of/from the cultivation of the EL4-B5 cells of the        previous step, i.e. from the cultivation of EL4-B5 cells that        has a cell density of from about 600,000 cells/ml to about        1,500,000 cells/ml.

In one embodiment the cultivation of said EL4-B5 cells is to a (final)cell density of from about 650,000 cells /ml to about 1,450,000cells/ml.

In one embodiment the cultivation of said EL4-B5 cells is to a (final)cell density of from about 650,000 cells/ml to about 825,000 cells/ml.

In one embodiment the cultivation of said EL4-B5 cells is to a (final)cell density of from about 1,400,000 cells/ml to about 1,500,000cells/ml.

In one embodiment of all aspects the EL4-B5 cells are irradiated withγ-radiation prior to the addition to the one or more B-cells. The doseof the γ-radiation is a sub-lethal dose. In one embodiment the EL4-B5cells are harvested when the cultivation of said EL4-B5 has a celldensity of from about 600,000 cells/ml to about 1,500,000 cells/ml, thecell density in the harvest is adjusted to a cell density of about 1×10⁶cells/ml, the cell density adjusted EL4-B5 cell suspension is irradiatedwith γ-radiation of a dose of 50 Gy, and a respective aliquot of theirradiated EL4-B5 cells is added to the one or more B-cells. In oneembodiment the aliquot is about 10⁴ to 10⁵ EL-4-B5 cells per B-cell.

In one embodiment of all aspects the one or more B-cells are incubatedwith about 10⁴ to 10⁵ EL4-B5 cells per B-cell. In one embodiment of allaspects the one or more B-cells are incubated with about 1×10⁴ to about5×10⁴ EL4-B5 cells per B-cell. In one embodiment of all aspects the oneor more B-cells are incubated with about 2×10⁴ EL4-B5 cells per B-cell.Thus the aliquot of the cultivation of the EL4-B5 cells that is added tothe one or more B-cells is about 10⁴ to 10⁵, or about 1×10⁴ to about5×10⁴, or about 2×10⁴ EL4-B5 cells per B-cell.

In one embodiment of all aspects the incubating is additionally in thepresence of a feeder mix.

In one embodiment the feeder mix comprises one or more of

-   -   i) interleukin-1 beta and tumor necrosis factor alpha,    -   ii) interleukin-2 (IL-2) and/or interleukin-10 (IL-10),    -   iii) Staphylococcus aureus strain Cowan's cells (SAC),    -   iv) interleukin-21 (IL-21) and optionally interleukine-2 (IL-2),    -   v) B-cell activation factor of the tumor necrosis factor family        (BAFF),    -   vi) interleukin-6 (IL-6),    -   vii) interleukin-4 (IL-4), and    -   viii) thymocyte cultivation supernatant.

In one embodiment the feeder mix comprises Staphylococcus aureus strainCowan's cells (SAC) and thymocyte cultivation supernatant.

In one embodiment of all aspects the method is for the co-cultivation ofone B-cell.

In one preferred embodiment the one B-cell is a single deposited B-cell.

In one embodiment of all aspects the incubating is for 5 to 14 days.

One aspect as reported herein is a method for producing an antibodycomprising the co-cultivation method as reported herein and therebyproducing an antibody.

All aspects (methods and uses) as reported herein comprise the step of

-   -   (individually) co-cultivating/incubating (each single deposited        or a pool of) B-cell(s) with feeder cells in a co-cultivation        medium, which has been supplemented with a feeder mix.

The result of the co-cultivation is a B-cell clone, i.e. a population ofB-cells that are the progeny of a single B-cell.

In one embodiment the methods as reported herein comprise prior to theco-cultivating step the following step:

-   -   depositing those B-cells of a population of B-cells that have        been labeled with one to three or one to four fluorescence        dyes/fluorophores as single cells.

In one embodiment the methods as reported herein comprise prior to theco-cultivating step the following step:

-   -   depositing those B-cells of a population of B-cells as single        cells that have been contacted with one to four antibodies each        specifically binding to a different B-cell surface antigen,        whereby each antibody is conjugated to a different fluorescent        dye, but labeled only with one to four fluorescence dyes.

The labeling is in one embodiment by contacting the B-cell population(sequentially or simultaneously) with one to four fluorescently labeledantibodies. Thereby a labeled B-cell preparation is obtained. Each ofthe fluorescently labeled antibodies binds to a different B-cell surfacemarker/target.

The depositing is by introducing the labeled B-cell preparation into aflow cytometer and depositing those cells as single cells that have beenlabeled with one to four fluorescent labels. As it is possible toincubate the cells with more fluorescent dyes as those which are usedfor selecting the cells in the cell sorter the cells can be selected forthe presence of specific surface markers and (optionally) simultaneouslyfor the absence of other surface markers.

The labeling and single cell deposition is done in order to reduce thecomplexity of the B-cell population by depleting those B-cells that arenot likely to produce an antibody having the intended characteristics.The labeled antibodies bind to a specific polypeptide displayed on thesurface of B-cells and, thus, provide for a positive selection label.Likewise it is also possible to select cells that are only labeled witha reduced number of fluorescent dyes compared to the number of labeledantibodies with which the B-cell had been incubated, such as e.g. cellshaving one fluorescent label out of two (i.e. incubation with twofluorescently label antibodies has been performed but only one thereofbinds to the B-cells), or two out of three. Based on thebinding/non-binding of the fluorescently labeled antibodies to theindividual B-cells of the B-cell population it is possible to identifyand separate target B-cells using a microfluidic sorting apparatus.Concomitantly with the selection also the amount of the label can bedetermined.

In one embodiment the methods as reported herein comprise the step ofincubating the population of B-cells in the co-cultivation medium priorto the single cell depositing/deposition. In one embodiment theincubating is at about 37° C. In one embodiment the incubating is for0.5 to two hours. In one embodiment the incubating is for about onehour. In one preferred embodiment the incubating is at about 37° C. forabout one hour.

In one embodiment the methods as reported herein comprise after thedepositing step and before the co-cultivating step the step ofcentrifuging the single cell deposited B-cells. In one embodiment thecentrifuging is for about 1 min. to about 30 min. In one embodiment thecentrifuging is for about 5 min. In one embodiment the centrifuging isat about 100×g to about 1,000×g. In one embodiment the centrifuging isat about 300×g. In one preferred embodiment the centrifuging is forabout 5 min. at about 300×g.

In one embodiment the method for selecting/obtaining a B-cell (clone)comprises the following steps:

-   -   a) labeling the B-cells of a population of B-cells with one to        four fluorescent dyes (optionally by incubating the B-cell        population with two to four fluorescently labeled antibodies        specifically binding to two to four different pre-determined        B-cell surface markers),    -   b) optionally incubating the cells in co-cultivation medium,    -   c) depositing those B-cells of the population of B-cells that        have been labeled with one to four fluorescent dyes (and        optionally not labeled with the other fluorescent dye(s)) as        single cells onto preloaded feeder cells,    -   d) optionally centrifuging the single deposited B-cells with the        preloaded feeder cells,    -   e) (individually) co-cultivating each single deposited B-cell        with feeder cells in a co-cultivation medium, which has been        supplemented with a feeder mix,    -   f) selecting a B-cell clone proliferating and secreting an        antibody in step e).

In one embodiment the method for producing an antibody specificallybinding to a target comprises the following steps

-   -   a) labeling the B-cells of a population of B-cells with one to        four fluorescent dyes (optionally by incubating the B-cell        population with two to four fluorescently labeled antibodies        specifically binding to two to four different pre-determined        B-cell surface markers),    -   b) optionally incubating the cells in co-cultivation medium,    -   c) depositing those B-cells of the population of B-cells that        have been labeled with one to four fluorescent dyes (and        optionally not labeled with the other fluorescent dye(s)) as        single cells onto preloaded feeder cells,    -   d) optionally centrifuging the single deposited B-cells with the        preloaded feeder cells,    -   e) (individually) co-cultivating each single deposited B-cell        with feeder cells in a co-cultivation medium, which has been        supplemented with a feeder mix,    -   f) selecting a B-cell clone of step e) secreting an antibody,    -   g) i) obtaining one or more nucleic acids encoding the secreted        antibody's variable domains from the B-cell clone selected in        step f),        -   ii) if the B-cell clone is not a human B-cell clone            humanizing the variable domains and providing the respective            encoding nucleic acids, and        -   iii) introducing the one or more nucleic acids in one or            more expression vectors,    -   h) cultivating a cell, which has been transfected with the one        or more expression vectors of step g), and recovering the        antibody from the cell or the cultivation supernatant and        thereby producing the antibody.

In one embodiment the method for producing an antibody comprising thefollowing steps

-   -   a) labeling the B-cells of a population of B-cells with one to        four fluorescent dyes (optionally by incubating the B-cell        population with two to four fluorescently labeled antibodies        specifically binding to two to four different pre-determined        B-cell surface markers),    -   b) optionally incubating the cells in co-cultivation medium,    -   c) depositing those B-cells of a population of B-cells that have        been labeled with one to four fluorescent dyes (and optionally        not labeled with the other fluorescent dye(s)) as single cells        onto preloaded feeder cells,    -   d) optionally centrifuging the single deposited B-cells with the        preloaded feeder cells,    -   e) (individually) co-cultivating each single deposited B-cell        with feeder cells in a co-cultivation medium, which has been        supplemented with a feeder mix,

f) determining the binding specificity of the antibodies secreted in thecultivation medium of the individual B-cells,

-   -   g) obtaining one or more nucleic acids encoding the secreted        antibody's variable domains from the B-cell clone by a reverse        transcriptase PCR and nucleotide sequencing, (and thereby        obtaining a monoclonal antibody variable light and heavy chain        domain encoding nucleic acid,)    -   h) if the B-cell is a non-human B-cell humanizing the variable        light and heavy chain domain and providing a nucleic acid        encoding the humanized variable domains,    -   i) introducing the monoclonal antibody variable light and heavy        chain variable domain encoding nucleic acid in one or more        expression vectors for the expression of an (human or humanized)        antibody,    -   j) introducing the expression vector(s) in a cell,    -   k) cultivating the cell and recovering the antibody from the        cell or the cell culture supernatant and thereby producing the        antibody.

In one embodiment the obtaining one or more nucleic acids encoding thesecreted antibody's variable domains from the B-cell clone comprises thefollowing steps:

-   -   extracting total RNA from the antibody-producing B-cell clone,    -   performing a single stranded cDNA synthesis/reverse        transcription of the extracted polyA⁺ mRNA,    -   performing a PCR with a set of species specific primer,    -   optionally removal of the PCR primer/purification of the PCR        product,    -   optionally sequencing of the PCR product.

In one embodiment the introducing the monoclonal antibody variable lightand/or heavy chain variable domain encoding nucleic acid in anexpression vector for the expression of an (human or humanized) antibodycomprises the following steps:

-   -   T4 polymerase incubation of the variable light and heavy chain        variable domain,    -   linearization and amplification of the expression vector,    -   T4 polymerase incubation of the amplified expression vector,    -   sequence and ligation independent cloning of the variable domain        encoding nucleic acid into the amplified expression vector, and    -   preparation of the vector(s) from pool of vector transformed E.        coli cells.

In one embodiment of all aspects the method comprises immediately priorto the labeling step the following step:

-   -   incubating the population of B-cells with (target) antigen,        which is immobilized on a solid surface, and recovering (only)        B-cells bound to the immobilized antigen.

In one embodiment of all aspects the population of B-cells is anon-human animal B-cell population. In one embodiment the B-cellpopulation is a mouse B-cell population, or a hamster B-cell population,or a rabbit B-cell population. In one preferred embodiment the B-cellpopulation is a rabbit B-cell population.

In one embodiment of all aspects the population of B-cells is obtainedfrom the blood of a non-human animal 4 days after the immunization. Inone embodiment the population of B-cells is obtained from the blood of anon-human animal of from 4 days up to at most 13 days afterimmunization.

In one embodiment the B-cell population is a human B-cell population.

In one embodiment of all aspects the population of B-cells is obtainedfrom blood by density gradient centrifugation.

In one embodiment of all aspects the B-cells are mature B-cells.

In one embodiment of all aspects the single cells are deposited(individually) into the wells of a multi-well plate.

In one embodiment of all aspects the feeder mix is natural thymocytecultivation supernatant (TSN) or a synthetic feeder mix. In oneembodiment the thymocyte cultivation supernatant is obtained fromthymocytes of the thymus gland of a young animal.

In one embodiment of all aspects the feeder mix is a synthetic feedermix. In one embodiment the synthetic feeder mix comprises p1 i)interleukin-1 beta and tumor necrosis factor alpha, and/or

-   -   ii) interleukin-2 (IL-2) and/or interleukin-10 (IL-10), and/or    -   iii) Staphylococcus aureus strain Cowan's cells (SAC), and/or    -   iv) interleukin-2 1 (IL-21) and optionally interleukine-2        (IL-2), and/or    -   v) B-cell activation factor of the tumor necrosis factor family        (BAFF), and/or    -   vi) interleukin-6 (IL-6), and/or p1 vii) interleukin-4 (IL-4).

In one embodiment of all aspects the feeder cells are murine EL-4 B5cells.

In one embodiment the feeder cells are murine EL-4 B5 cells and thefeeder mix comprises IL-1β, TNF-α, IL-10 and one or more selected fromIL-21, SAC, BAFF, IL-2, IL-4, and IL-6.

In one embodiment the feeder cells are murine EL-4 B5 cells and thefeeder mix comprises IL-1β, TNF-α, IL-10, SAC and IL-2.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the feeder cells are murine EL-4 B5 cells andfeeder mix is thymocyte cultivation supernatant.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the feeder cells are murine EL-4 B5 cells and thefeeder mix is consisting of IL-1β, TNF-α, and any two of IL-2, IL-6 andIL-10.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the feeder cells are murine EL-4 B5 cells and thefeeder mix is consisting of IL-1β, TNF-α, IL-6 and IL-10.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the feeder cells are murine EL-4 B5 cells and thefeeder mix comprises IL-1β, TNF-α, IL-10, SAC and IL-2 or IL-6.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the feeder cells are murine EL-4 B5 cells and thefeeder mix comprises IL-1β, TNF-α, IL-21 and at least one of IL-2, IL-10and IL-6.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the feeder cells are rabbit CD40L-expressing CHOcells. In one embodiment the feeder mix comprises IL-2 and IL-21 andoptionally IL-6.

In one embodiment of all aspects the antibody is a monoclonal antibody.

In one embodiment of all aspects the deposited cells are labeled withone or four fluorescence dyes and the incubation is with two to fourfluorescently labeled antibodies.

In one embodiment of all aspects the labeling of the B-cells of thepopulation of B-cells results in labeling of 0.1% to 2.5% of the cellsof the (total) B-cell population.

In one embodiment of all aspects the labeling is of B-cell surface IgG.

In one preferred embodiment of all aspects the incubation is with afluorescently labeled anti-IgG antibody and a fluorescently labeledanti-IgM antibody (the labeling is of cell surface IgG and cell surfaceIgM) and the selection is of cells positive for cell surface IgG andnegative for cell surface IgM (results in single cell deposition ofIgG⁺IgM⁻-B-cells).

In one preferred embodiment of all aspects the incubation is with afluorescently labeled anti-IgG antibody and a fluorescently labeledanti-IgM antibody (the labeling is of cell surface IgG and cell surfaceIgM) and the selection is of cells positive for cell surface IgG andnegative for cell surface IgM (results in single cell deposition ofIgG⁺IgM⁻-B-cells), whereby the population of B-cells has been incubatedwith (target) antigen, which is immobilized on a solid surface, and(only) B-cells bound to the immobilized antigen have been recovered andsubjected to the incubation with the fluorescently labeled antibodies.

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the incubation is with a fluorescently labeledanti-IgG antibody (the labeling is of cell surface IgG) and theselection is of cells positive for cell surface IgG (results in singlecell deposition of IgG⁺-B-cells).

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the incubation is with a fluorescently labeledanti-CD138 antibody (the labeling is of cell surface CD138) and theselection is of cells positive for cell surface CD138 (results in singlecell deposition of CD138+-B-cells).

In one embodiment of all aspects the B-cell population is a rabbitB-cell population and the incubation is with a fluorescently labeledanti-IgG antibody and a fluorescently labeled anti-CD138 antibody (thelabeling is of cell surface IgG and cell surface CD138) and theselection is of cells positive for cell surface IgG and also positivefor cell surface CD138 (results in single cell deposition ofIgG⁺CD138⁺-B-cells).

In one preferred embodiment of all aspects the B-cell population is arabbit B-cell population and the incubation is with a fluorescentlylabeled anti-IgG antibody, a fluorescently labeled anti-IgM antibody andoptionally a fluorescently labelled anti-human IgG light chain antibodyin case of human transgenic rabbit (the labeling is of cell surface IgG,cell surface IgM and optionally cell surface human IgG light chain) andthe selection is of cells positive for cell surface IgG, negative forcell surface IgM and optionally positive for cell surface human IgGlight chain (results in single cell deposition of IgG⁺IgM⁻-B-cells(optionally IgG⁺IgM⁻huIgGLC⁺-B-cells).

In one embodiment of all aspects the co-cultivating is in aco-cultivation medium comprising RPMI 1640 medium supplemented with 10%(v/v) FCS, 1% (w/v) of a 200 mM glutamine solution that comprisespenicillin and streptomycin, 2% (v/v) of a 100 mM sodium pyruvatesolution, and 1% (v/v) of a 1 M2-(4-(2-hydroxyethyl)-1-piperazine)-ethane sulfonic acid (HEPES) buffer.In one embodiment the co-cultivation medium further comprises 0.05 mMbeta-mercaptoethanol.

In one embodiment the animal is an experimental animal. In oneembodiment the experimental animal is selected from mouse, hamster, andrabbit. In one embodiment the experimental animal is a rabbit.

DESCRIPTION OF THE FIGURES

FIG. 1 Average frequency of antigen-specific IgG positive wells per IgGpositive wells.

FIG. 2 Average frequency of antigen-specific IgG positive wells per IgGpositive wells.

DEFINITIONS

“Affinity” refers to the strength of the total sum of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

The term “amino acid” as used within this application denotes the groupof carboxy α-amino acids, which directly or in form of a precursor canbe encoded by a nucleic acid. The individual amino acids are encoded bynucleic acids consisting of three nucleotides, so called codons orbase-triplets. Each amino acid is encoded by at least one codon. This isknown as “degeneration of the genetic code”. The term “amino acid” asused within this application denotes the naturally occurring carboxyα-amino acids comprising alanine (three letter code: ala, one lettercode: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp,D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu,L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F),proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp,W), tyrosine (tyr, Y), and valine (val, V).

The term “antibody” herein is used to denote naturally occurringantibodies including their naturally occurring structural variants.

For example, native (human, mouse, rat, rabbit) IgG antibodies areheterotetrameric glycoproteins with a molecular weight of about 150,000Dalton. Native IgG antibodies are composed of two identical light chainsand two identical heavy chains comprising inter- and intra-chaindisulfide bonds, so that all four chains are covalently linked to eachother. From N- to C-terminus, each heavy chain has a variable region(VH), also called a variable heavy chain domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),whereby a flexible hinge region is located between the first and thesecond constant domain. The heavy chain of an antibody may be assignedto one of five types, called IgA, IgD, IgE, IgG and IgM, depending ontheir sequence and domain structure (“class” of an antibody). Several ofthese may be further divided into subclasses (isotypes), e.g., IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively. Similarly, from N- to C-terminus, each lightchain has a variable region (VL), also called a variable light chaindomain or a light chain variable domain, followed by a constant lightchain domain (CL). The light chain of an antibody may be assigned to oneof two types, called kappa (κ) and lambda (λ), based on the amino acidsequence of its constant domain.

For example, native (camelid, i.e. from Camelidae, sub-order Tylopoda,which includes camels, dromedaries and llamas) heavy-chain onlyantibodies (VHH antibodies) do not comprise a classical CH1 domain asfound in conventional IgG heavy chains, and, thus, are expressed as VHHdomains fused directly to the hinge-CH2-CH3 domains of an antibody. Thevariable region sequences from llama derived VHH antibodies, forexample, are similar to sequences in the human VH3 family of variabledomains (Schroeder et al., Int. Immunol. 2 (1989) 41-50). Compared toantibodies of the IgG type the CDR3 domain amino acid sequence in L.llama VHH domains is longer on average than most CDR3 domains ofclassical IgG type antibodies comprising heavy and light chains. Alikeclassical IgG antibodies the position of the CDRs in VHH antibodies canbe determined by methods well known in the art (see e.g. U.S. Pat. No.5,637,677). Residues 11, 37, 44, 45 and 47 are important for theformation of the chain interface (see e.g. WO 99/42077).

An “antibody fragment” refers to a molecule other than an intactantibodies (IgG/VHH=four chain/two chain) comprising only a portion ofan intact antibody and that binds to the same antigen to which theintact antibody binds. Examples of antibody fragments include but arenot limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linearantibodies; single-chain antibody molecules (e.g. scFv); single domainantibodies; and multispecific antibodies formed from antibody fragments.

The term “cell” includes both prokaryotic cells, which are used forpropagation of plasmids, and eukaryotic cells, which are used for theexpression of a nucleic acid. In one embodiment the eukaryotic cell is amammalian cell. In one embodiment the mammalian cell is a CHO cell,optionally a CHO K1 cell (e.g. a ATCC CCL-61 or DSM ACC 110), or a CHODG44 cell (also known as CHO-DHFR[-], e.g. a DSM ACC 126), or a CHO XL99cell, a CHO-T cell (see e.g. Morgan, D., et al., Biochemistry 26 (1987)2959-2963), or a CHO-S cell, or a Super-CHO cell (Pak, S.C.O., et al.Cytotechnol. 22 (1996) 139-146), or BHK cell, or a NSO cell, or a Sp2/0cell, or a HEK 293 cell, or a HEK 293 EBNA cell, or a PER.C6® cell, or aCOS cell. If these cells are not adapted to growth in serum-free mediumor in suspension an adaptation prior to the use in the current methodcan be performed. As used herein, the expression “cell” includes thesubject cell and its progeny. Thus, the words “transformant” and“transformed cell” include the primary subject cell and cultures derivedthere from without regard for the number of transfers orsub-cultivations. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

The term “clone” denotes a population of dividing and antibody secretingB-cells arising from/originating from a single B-cell. Thus, a B-cellclone is a homogeneous population of B-cells and produces a monoclonalantibody.

The term “cognate pair of antibody variable domains” denotes a pair ofantibody variable domains that is obtained from a single antibodysecreting B-cell (clone), i.e. which has been generated as pair duringthe immune response of a mammal due to the contact with an immunogenicmolecule or which have been assembled randomly during a displayapproach.

The term “experimental animal” denotes a non-human animal. In oneembodiment the experimental animal is selected from rat, mouse, hamster,rabbit, camel, llama, non-human primates, sheep, dog, cow, chicken,amphibians, sharks and reptiles. In one embodiment the experimentalanimal is a rabbit.

The term “expression” as used herein refers to transcription and/ortranslation and secretion processes occurring within a cell. The levelof transcription of a nucleic acid sequence of interest in a cell can bedetermined on the basis of the amount of corresponding mRNA that ispresent in the cell. For example, mRNA transcribed from a sequence ofinterest can be quantified by qPCR or RT-PCR or by Northernhybridization (see Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)). Polypeptides encoded by a nucleic acid can bequantified by various methods, e.g. by ELISA, by assaying the biologicalactivity of the polypeptide, or by employing assays that are independentof such activity, such as Western blotting or radioimmunoassay, usingimmunoglobulins that recognize and bind to the polypeptide (seeSambrook, et al., (1989), supra).

To a person skilled in the art procedures and methods are well known toconvert an amino acid sequence, e.g. of a polypeptide, into acorresponding nucleic acid sequence encoding this amino acid sequenceand vice versa. Therefore, a nucleic acid is characterized by itsnucleic acid sequence consisting of individual nucleotides and likewiseby the amino acid sequence of a polypeptide encoded thereby.

An “expression cassette” denotes a construct that contains the necessaryregulatory elements, such as promoter and polyadenylation site, forexpression of at least the contained nucleic acid in a cell.

Expression can be performed either as transient expression or a stableexpression. Antibodies are in general secreted into the cultivationmedium by the cell producing it. Therefore non-mature antibody chainscontain an N-terminal extension (also known as the signal sequence),which is necessary for the transport/secretion of the antibody throughthe cell wall into the extracellular medium. In general, the signalsequence for recombinant production of an antibody can be derived fromany gene encoding a secreted polypeptide. If a heterologous signalsequence is used, it should be one that is recognized and processed(i.e. cleaved by a signal peptidase) by the host cell. For secretion inyeast for example the native signal sequence of a heterologous gene tobe expressed may be substituted by a homologous yeast signal sequencederived from a secreted gene, such as the yeast invertase signalsequence, alpha-factor leader (including Saccharomyces, Kluyveromyces,Pichia, and Hansenula α-factor leaders, the second described in U.S.Pat. No. 5,010,182), acid phosphatase signal sequence, or the C.albicans glucoamylase signal sequence (EP 0 362 179). In mammalian cellsthe native signal sequence is satisfactory, although other mammaliansignal sequences may be suitable, such as signal sequences from othersecreted polypeptides of the same or related species as well as viralsecretory signal sequences, for example, the herpes simplex glycoproteinD signal sequence. The DNA fragment encoding for such a pre-segment isligated in frame, i.e. operably linked, to the DNA fragment encoding anantibody chain.

The term “expression machinery” denotes the sum of the enzymes,cofactors, etc. of a cell that is involved in the steps of geneexpression beginning with the transcription step of a nucleic acid orgene (i.e. also called “gene expression machinery”) to thepost-translational modification of the polypeptide encoded by thenucleic acid. The expression machinery e.g. comprises the steps oftranscription of DNA into pre-mRNA, pre-mRNA splicing to mature mRNA,translation into a polypeptide of the mRNA, and post translationalmodification of the polypeptide.

An “expression plasmid” or “expression vector” is a nucleic acidproviding all required elements for the expression of the comprisedstructural gene(s) in a host cell. Typically, an expressionplasmid/vector comprises a prokaryotic plasmid propagation unit, e.g.for E. coli, comprising an origin of replication, and a selectablemarker, a eukaryotic selection marker, and one or more expressioncassettes for the expression of the structural gene(s) of interest eachcomprising a promoter, a structural gene, optionally a transcriptionterminator and a polyadenylation signal. Gene expression is usuallyplaced under the control of a promoter, and such a structural gene issaid to be “operably linked to” the promoter. Similarly, a regulatoryelement and a core promoter are operably linked if the regulatoryelement modulates the activity of the core promoter.

The term “feeder mix” denotes a combination of different additives, suchas growth factors, cytokines and/or further proteins promoting theactivation and/or survival of B-cells and/or antibody secretion. Thefeeder mix can be a natural feeder mix, e.g. obtained from thecultivation supernatant of thymocytes (TSN), which is a non-definedcombination of cytokines. Alternatively the feeder mix can be asynthetic feeder mix, which is a defined combination of differentrecombinantly produced or chemically synthesized additives, such asgrowth factors, cytokines and/or further proteins promoting theactivation and/or survival of B-cells and/or antibody secretion.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” or “transfectants” and “transformed cells”and “transfected cells”, which include the primary transformed cell andprogeny derived therefrom without regard to the number of passages.Progeny may not be completely identical in nucleic acid content to aparent cell, but may contain mutations. Mutant progeny that have thesame function or biological activity as screened or selected for in theoriginally transformed cell are included herein.

A “human antibody” is an antibody, which possesses an amino acidsequence that corresponds to that of an antibody produced by a human ora human cell or derived from a non-human source that utilizes humanantibody repertoires or other human antibody-encoding sequences. Thisdefinition of a human antibody specifically excludes a humanizedantibody comprising non-human antigen-binding residues.

An “individual” or “subject” is a vertebrate. In one embodiment thevertebrate is a mammal. Mammals include, but are not limited to,domesticated animals (e.g., cows, sheep, cats, dogs, and horses),primates (e.g., humans and non-human primates such as monkeys), rabbits,and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human. In other embodiments the individual orsubject is a rabbit.

The term “labeling” denotes a process for determining the presence orabsence of a surface marker, which can be determined bybinding/non-binding of a specifically binding and labeled anti-surfacemarker antibody to a cell. Thus, the presence of a surface marker isdetermined e.g. in the case of a fluorescence label by the occurrence ofa fluorescence whereas the absence of a surface marker is determined bythe absence of a fluorescence after incubation of a cell or a populationof cells with the respective specifically binding and labeledanti-surface marker antibody.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodiesproduced by a single cell clone, i.e., the individual antibodiescomprising the population are identical and/or bind the same epitope,except for possible variant antibodies, e.g., containing naturallyoccurring mutations or arising during production of a monoclonalantibody preparation, such variants generally being present in minoramounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

For example, the monoclonal antibodies to be used in accordance with thepresent invention may be made by a variety of techniques, including butnot limited to methods utilizing transgenic animals containing all orpart of the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The terms “nucleic acid” and “nucleic acid sequence” denote a polymericmolecule consisting of individual nucleotides (also called bases) a, c,g, and t (or u in RNA), for example to DNA, RNA, or modificationsthereof. This polynucleotide molecule can be a naturally occurringpolynucleotide molecule or a synthetic polynucleotide molecule or acombination of one or more naturally occurring polynucleotide moleculeswith one or more synthetic polynucleotide molecules. Also encompassed bythis definition are naturally occurring polynucleotide molecules inwhich one or more nucleotides are changed (e.g. by mutagenesis),deleted, or added. A nucleic acid can either be isolated, or integratedin another nucleic acid, e.g. in an expression cassette, aplasmid/vector, or the chromosome of a host cell. A nucleic acid ischaracterized by its nucleic acid sequence consisting of individualnucleotides.

“Operably linked” refers to a juxtaposition of two or more components,wherein the components so described are in a relationship permittingthem to function in their intended manner. For example, a promoterand/or enhancer are operably linked to a coding sequence, if it acts incis to control or modulate the transcription of the linked sequence.Generally, but not necessarily, the DNA sequences that are “operablylinked” are contiguous and, where necessary to join two protein encodingregions such as a secretory leader and a polypeptide, contiguous and in(reading) frame. However, although an operably linked promoter isgenerally located upstream of the coding sequence, it is not necessarilycontiguous with it. Enhancers do not have to be contiguous. An enhanceris operably linked to a coding sequence if the enhancer increasestranscription of the coding sequence. Operably linked enhancers can belocated upstream, within or downstream of coding sequences and atconsiderable distance from the promoter. A polyadenylation site isoperably linked to a coding sequence if it is located at the downstreamend of the coding sequence such that transcription proceeds through thecoding sequence into the polyadenylation sequence. A translation stopcodon is operably linked to an exonic nucleic acid sequence if it islocated at the downstream end (3′ end) of the coding sequence such thattranslation proceeds through the coding sequence to the stop codon andis terminated there. Linking is accomplished by recombinant methodsknown in the art, e.g., using PCR methodology and/or by ligation atconvenient restriction sites. If convenient restriction sites do notexist, then synthetic oligonucleotide adaptors or linkers are used inaccord with conventional practice.

A “structural gene” denotes the region of a gene without a signalsequence, i.e. the coding region.

The term “specifically binding” and grammatical equivalents thereofdenote that the antibody binds to its target with a dissociationconstant (KD) of 10⁻⁷M or less, in one embodiment of from 10⁻⁸ M to10⁻¹³ M, in a further embodiment of from 10⁻⁹ M to 10⁻¹³ M. The term isfurther used to indicate that the antibody does not specifically bind toother biomolecules present, i.e. it binds to other biomolecules with adissociation constant (KD) of 10⁻⁶M or more, in one embodiment of from10⁻⁶M to 1 M.

A “transfection plasmid/vector” is a nucleic acid (also denoted asnucleic acid molecule) providing all required elements for theexpression of the in the transfection plasmid/vector comprised codingnucleic acids/structural gene(s) in a host cell. A transfectionplasmid/vector comprises a prokaryotic plasmid propagation unit, e.g.for E. coli, in turn comprising a prokaryotic origin of replication, anda nucleic acid conferring resistance to a prokaryotic selection agent,further comprises the transfection plasmid/vector one or more nucleicacid(s) conferring resistance to an eukaryotic selection agent, and oneor more nucleic acid encoding a polypeptide of interest. The nucleicacids conferring resistance to a selection agent and the nucleic acid(s)encoding a polypeptide of interest are placed each within an expressioncassette, whereby each expression cassette comprises a promoter, acoding nucleic acid, and a transcription terminator including apolyadenylation signal. Gene expression is usually placed under thecontrol of a promoter, and such a structural gene is said to be“operably linked to” the promoter. Similarly, a regulatory element and acore promoter are operably linked if the regulatory element modulatesthe activity of the core promoter.

The term “variable region” or “variable domain” refers to the region ofan antibody heavy or light chain that is involved in the binding of theantibody to its antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs) (see,e.g., Kindt, T. J., et al., Kuby Immunology, 6th ed., W.H. Freeman andCo., N.Y. (2007), page 91). A single VH or VL domain may be sufficientto confer antigen-binding specificity. Furthermore, antibodies that binda particular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively (see, e.g., Portolano, S., et al., J.Immunol. 150 (1993) 880-887; Clackson, T., et al., Nature 352 (1991)624-628).

The term “young animal” denotes an animal before sexual maturity occurs.A young hamster, for example, is of an age of less than 6 weeks,especially less than 4 weeks. A young mouse, for example, is of an ageof less than 8 weeks, especially less than 5 weeks.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is based at least in part on the finding that thecell density of the cultivation from which the EL4-B5 feeder cells thatare used in a co-cultivation with B-cells are taken has an influence onthe frequency (i.e. relative number) of B-cells clones obtained in theco-cultivation of B-cells and EL-4-B5 feeder cells that secrete anantigen-specific antibody. This finding is surprising as the art teachesthat a maximum cell density of EL4-B5 cells of 1,000,000 cells/ml insole EL-4 B5 cultivations shall not be exceeded. It has been found thatthis is not the case and that even higher cell densities can be usedwhich allows for a more efficient production of the EL4-B5 cells, i.e.the number of cultivations is reduced as a higher cell density can beused without changing the number of EL4-B5 cells employed in theco-cultivation.

The current invention is based at least in part on the finding thatoverall the frequency of antigen binding and IgG positive wells amongstall IgG positive wells increases when the EL-4 B5 cells used in theco-cultivation have been obtained from a cultivation with EL-4 B5 celldensities higher than published (i.e. higher than 500,000 cell/ml) atthe time of harvest of the EL-4-B5 cells are employed. This effect wasobserved up to a cell density of 1,500,000 cells/ml.

Immunization

For the generation of therapeutic antibodies either a non-human animalis immunized with the therapeutic target (either alone or in combinationwith an immunogenic stimulus) to elicit an immune response or syntheticapproaches, such as phage display libraries are used. If a transgenicanimal (i.e. having a human immune system) or a human phage displaylibrary is used human antibodies are obtained.

Otherwise non-human animal antibodies are obtained that will behumanized thereafter. A rare possibility to obtain potential therapeuticantibodies is from the blood of a human being that has recovered from adisease.

Often non-human animals, such as mice, rabbits, hamster and rats, areused as animal model for evaluating antibody based therapies. Therefore,it is normally required to provide cross-reactive antibodies binding tothe non-human animal antigen as well as to the human antigen.

In the method as reported herein B-cells obtained from any source e.g.human, mouse, hamster or rabbit, can be used. Depending on the source ofthe B-cell the feeder cells and the feeder mix are adjusted/chosen.

In case of a rabbit B-cell the feeder cell is either an EL4-B5 cell or amammalian cell, such as a CHO cell or a BHK cell or a HEK cell,expressing rabbit CD40L. In one embodiment the rabbit is selected fromNew Zealand White (NZW) rabbits, Zimmermann-rabbits (ZIKA),Alicia-mutant strain rabbits, basilea mutant strain rabbits, transgenicrabbits with a human immunoglobulin locus, rbIgM knock-out rabbits, andcross-breeding thereof.

In case of a human B-cell the feeder cell is either an EL4-B5 cell or amammalian cell, such as a CHO cell or a BHK cell or a HEK cell,expressing human CD40L.

In case of a murine B-cell the feeder cell is either an EL4-B5 cell or amammalian cell, such as a CHO cell or a BHK cell or a HEK cell,expressing mouse CD40L. In one embodiment the mouse is an NMRI-mouse ora balb/c-mouse.

In case of a hamster B-cell the feeder cell is either an EL4-B5 cell ora mammalian cell, such as a CHO cell or a BHK cell or a HEK cell,expressing hamster CD40L. In one embodiment the hamster is selected fromArmenian hamster (Cricetulus migratorius), Chinese hamster (Cricetulusgriseus), and Syrian hamster (Mesocricetulus auratus). In a preferredembodiment the hamster is the Armenia hamster.

Source and Isolation of B-Cells

The blood provides a high diversity of antibody producing B-cells. Thetherefrom obtained B-cell clones secrete antibodies that have almost noidentical or overlapping amino acid sequences within the CDRs, thus,show a high diversity.

In one embodiment B-cells, e.g. from the blood, are obtained of from 4days after immunization until at most 14 days after immunization or themost recent boost of the non-human animal. This time span allows for ahigh flexibility in the method as reported herein. In this time span itis likely that the B-cells providing for the most affine antibodiesmigrate from spleen to blood (see e.g. Paus, D., et al., JEM 203 (2006)1081-1091; Smith, K. G. S., et al., The EMBO J. 16 (1997) 2996-3006;Wrammert, J., et al., Nature 453 (2008) 667-672).

B-cells from the blood, e.g. of a non-human animal or from human blood,may be obtained with any method known in the art. For example, densitygradient centrifugation (DGC) or red blood cell lysis (lysis) can beused. Density gradient centrifugation compared to hypotonic lysisprovides for a higher overall yield, i.e. number of B-cell clones.Additionally from the cells obtained by density gradient centrifugationa larger number of cells divides and grows in the co-cultivation step.Also the concentration of secreted antibody is higher compared to cellsobtained with a different method. Therefore, in one embodiment theproviding of a population of B-cells is by density gradientcentrifugation.

Alternatively the B-cells can be obtained from spleen or other secondaryimmunological organs like lymphnodes or Peyers Patches.

Selection Steps Prior to Co-Cultivation

B-cells producing antibodies that specifically bind an antigen can beenriched from peripheral blood mononuclear cells (PBMCs). Thus, in oneembodiment of all methods as reported herein the B-cell population isenriched from peripheral blood mononuclear cells (PBMCs).

In one embodiment of all methods as reported herein the PBMCs aredepleted of macrophages. This is especially advantageous for B-cells ofrabbit origin for the co-cultivation step.

Macrophages can be depleted from PBMCs by adhesion to the surface of thecell culture plate (see pre-incubation step).

Incubating the population of B-cells in co-cultivation medium prior tothe single cell depositing can increase the total number of antibodysecreting cells obtained after the single cell depositing compared to asingle cell depositing directly after the isolation and optionalenrichment of the population of B-cells from the blood of a non-humananimal (in one embodiment the non-human animal is a rabbit).Specifically the incubating is at about 37° C. for about one hour inEL-4 B5 medium, e.g. using a cell culture incubator.

In one embodiment of the methods as reported herein the cells are from aprotein-immunized animal and are depleted of macrophages prior to thelabeling.

Cells not producing an antibody binding the antigen or, likewise, cellsproducing an antibody binding to the antigen can be reduced or enriched,respectively, by using a panning approach. Therein the respectiveantigen is presented attached to a surface and cells binding thereto areselectively enriched in the cell population in case the bound cells areprocessed further, or reduced in the cell population in case the cellsremaining in solution are processed further.

The method as reported herein comprises in one embodiment prior to thesingle cell depositing a selecting step in which B-cells producingspecific and/or non-cross-reactive antibodies are selected based on cellsurface markers and fluorescence activated cell sorting/gating. In oneembodiment mature B-cells are sorted/enriched/selected. For selection ofB-cells from different non-human animal species different cell surfacemarkers can be used.

With the labeling of non-target cell populations and non-specificallybinding lymphocytes it is possible to selectively deplete these cells.In this depletion step only a partial depletion can be achieved. Albeitthe depletion is not quantitative it reduces or even eliminates thenumber of interfering cells in the succeeding fluorescence labeling ofthe remaining cells. By a single cell depositing of mature B-cells(memory B-cells, affinity matured plasmablasts and plasma cells) byfluorescence activated cell sorting using the labeling a higher numberof IgG⁺-wells/cell clones can be obtained in the co-cultivation step.

Different cell populations can be labeled by using different surfacemarkers such as CD3⁺-cells (T-cells), CD19⁺-cells (B-cells), IgM⁺-cells(mature naive B-cells), IgG⁺-cells (mature B-cells), CD38⁺-cells (e.g.plasmablasts), and IgG⁺CD38⁺-cells (pre-plasma cells).

Immuno-fluorescence labeling for selection of mature IgG⁺-B-cells, suchas memory B-cells, plasmablasts, and plasma cells, is available. For aselection or enrichment of B-cells the cells are either single labeled,or double labeled, or triple labeled.

TABLE Immuno-fluorescence labeling for the determination of maturemouse-(A-J), hamster-(K) and rabbit (L-N)-B-cells. B-cell sorting of B-fraction of all origin cells with viable cells (%) mouse IgG⁺CD19⁺  0.5± 0.2 n = 14 mouse IgG⁺CD38⁺ 0.8 ± 0.5 n = 9 mouse IgG⁺CD138⁺ 0.06 ±0.07 n = 6 mouse IgG⁻CD138⁺ 0.6 ± 0.5 n = 6 mouse IgG⁺CD27⁺ 0.1 ± 0.1 n= 8 mouse CD27⁺CD138⁺ 1.5 ± 0.5 n = 2 mouse CD27⁺IgG⁺CD3⁻ 0.10 ± 0.04 n= 3 mouse CD3⁻CD27⁺ 1.33 n = 1 mouse IgG⁺CD268⁺  0.8 n = 1 mouseCD38⁺CD3⁻ 12 ± 7 n = 2  hamster IgG⁺IgM⁻  0.6 ± 0.1 n = 15 rabbit IgG⁺ 0.6 ± 0.2, n = 5 rabbit IgG⁺IgM⁻  0.4 ± 0.2, n = 2 rabbit IgG⁺CD138⁺ 0.3 ± 0.1, n = 5

In one embodiment the methods comprise the step of depleting the B-cellpopulation of macrophages and enriching of B-cells of the B-cellpopulation secreting antibody specifically binding a target antigen.

Single Cell Depositing

The method as reported herein comprises the step of depositing theB-cells of a B-cell population as single cells. In one embodiment of allmethods as reported herein the depositing as single cells is byfluorescence activated cell sorting (FACS). The surface marker used forthe labeling required for the FACS single cell depositing can be withthe specific marker combination as outlined herein.

An additional centrifugation step after the single cell depositing andprior to the co-cultivation can increase the number of antibodysecreting cells and the amount of the secreted IgG.

In one embodiment of all methods as reported herein the method comprisesthe step of centrifuging the single deposited cells prior to theco-cultivation. In one preferred embodiment the centrifuging is for 5min. at 300×g.

Co-Cultivation

The single deposited B-cells are co-cultivated with murine EL-4-B5feeder cells in the presence of a feeder mix.

As outlined above an increase in the yield in the co-cultivation step(number of IgG⁺-wells/cell clones as well as IgG-concentration) and alsoan enrichment or isolation of mature IgG⁺-B-cell from PBMCs can beachieved by suitable immuno fluorescence labeling.

The immuno-fluorescence labeling used for B-cells obtained from theblood of an experimental non-human animal can also be used for thelabeling of B-cells obtained from the spleen and other immunologicalorgans of an experimental non-human animal, such as mouse, hamster andrabbit. For rabbit-blood derived B-cells 0.2% of IgG⁺-cells were foundafter depletion of macrophages. Peyer'sche plaques from rabbit showed0.4% of IgG⁺-cells and spleen showed 0.3% of IgG⁺-cells after depletionof macrophages.

With the methods as reported herein after about seven (7) days, i.e.after 5, 6, 7, or 8 days, especially after 7 or 8 days, ofco-cultivation antibody concentrations of from about 30 ng/ml up to 15ng/ml or more can be obtained (average value about 500 ng/ml). With thethereby provided amount of antibody a high number of different analysescan be performed in order to characterize the antibody, e.g. regardingbinding specificity, in more detail. With the improved characterizationof the antibody at this early stage in the screening/selection processit is possible to reduce the number of required nucleic acid isolationsand sequencing reactions that have to be performed. Additionally theB-cell clone provides an amount of mRNA encoding monoclonal light andheavy chain variable region allowing the use of degenerated PCR primerand obviates the requirement of highly specific primer. Also therequired number of PCR cycles is reduced. Thus, in one embodiment thereverse transcriptase PCR is with degenerated PCR primer for the lightand heavy chain variable domain.

The co-cultivation step with feeder cells can be preceded and alsosucceeded by a number of additional steps.

In one embodiment of all methods as reported herein the feeder mix is athymocyte cultivation supernatant. In a specific embodiment thethymocyte cultivation supernatant is obtained from the thymocytes of thethymus gland of the respective non-human animal.

Due to the origin of the feeder mix, which is derived from thesupernatant of cultivated thymocytes (thymocyte cultivationsupernatant—TSN), considerable batch to batch variations may occur.

In order to overcome this variability a synthetic feeder mix consistingof synthetic components can be employed.

A synthetic feeder mix consisting of IL-1β (interleukin-1 beta), TNF-α(tumor necrosis factor alpha), IL-2 (interleukin-2) and IL-10(interleukin-10) is known from Tucci, A., et al., J. Immunol. 148 (1992)2778-2784.

The B-cell-species-specific additives for the synthetic feeder mix canresult in increased amounts of secreted antibody by the respectiveB-cell clone.

Concomitantly highly producing cells contain more mRNA which in turnfacilitates the reverse transcription and sequencing of the encodingnucleic acid, e.g. with a redundant, non-specific primer set.

By the addition of SAC (Staphylococcus aureus strain Cowan's cells, asingle SAC lot was used) the number of antibody secreting B-cells andthe average

IgG-concentration in the supernatant after co-cultivation can beincreased. For the addition of SAC in the co-cultivation a concentrationrange can be defined as reduced as well as increased concentrations ofSAC reduce the amount of secreted antibody.

In one embodiment of all methods as reported herein the synthetic feedermix for the co-cultivation of murine B-cells comprises IL-1β, IL-2,IL-10, TNF-α and BAFF. In one embodiment BAFF is added at aconcentration of 5 ng/ml.

In one embodiment of all methods as reported herein the synthetic feedermix for the co-cultivation of hamster B-cells comprises IL-1β, IL-2,IL-10, TNF-α, IL-6 and SAC. In one embodiment IL-6 is added at aconcentration of 10 ng/ml. In one embodiment SAC is added at a 1:75,000ratio.

In the presence of EL-4 B5 feeder cells at least IL-1β and TNFα arerequired for the co-cultivation of mouse, hamster and rabbit B-cells.IL-2 and IL-10 can be omitted for the co-cultivation of murine cells.Hamster B-cells can be cultivated in the absence of either IL-2 orIL-10. Rabbit B-cells can be cultivated in the absence of either IL-2 orIL-10 or IL-6.

In one embodiment IL-1β, TNF-α, IL-2, IL-10 and IL-21 are recombinantmurine IL-1β, murine TNF-α, murine IL-2, murine IL-10, and murine IL-21.

In one embodiment BAFF is added at a concentration of 5 ng/ml.

In one embodiment IL-6 is added at a concentration of 10 ng/ml.

In one embodiment SAC is added at a 1:75,000 ratio.

The co-cultivation is in one embodiment of all methods as reportedherein in polystyrene multi well plates with wells with a round bottom.The working volume of the wells is in one embodiment of all methods asreported herein of 50 μl to 250 μl. In one embodiment the wells arecoated at least partially with a non-fibrous substrate prepared from ablend of polymer plastic resin and amphipathic molecules, wherein theamphipathic molecule comprises a hydrophilic moiety and a hydrophobicregion, wherein the hydrophobic regions are anchored within thesubstrate and the hydrophilic moieties are exposed on the substrate. Inone embodiment the amphipathic molecules are chosen from alkylamineethoxylated, poly (ethylene imine), octyldecamine or mixtures thereof(see e.g. EP 1 860 181).

Characterization of Co-Cultivated Cells

For the (qualitative and quantitative) determination of secreted IgGafter the co-cultivation generally all methods known to a person ofskill in the art such as an ELISA can be used. In one embodiment of allmethods as reported herein an ELISA is used.

Depending on the characterization results a B-cell clone can beobtained, i.e. selected. The term “clone” denotes a population ofdividing and antibody secreting

B-cells arising from/originating from a single B-cell. Thus, a B-cellclone produces a monoclonal antibody.

Isolation of mRNA, Cloning and Sequencing

From the B-cells the total mRNA can be isolated and transcribed in cDNA.With specific primers the cognate VH- and VL-region encoding nucleicacid can be amplified. Almost no identical sequences are obtained. Themethod provides for highly diverse antibodies binding to the sameantigen.

The primers used for the amplification of the VH-encoding nucleic acidcan be used for cDNA obtained from cells from the NMRI-mouse, theArmenian Hamster, the Balb/c-mouse as well as the Syrian hamster and therabbit.

In one embodiment of all methods as reported herein the amino acidsequence is derived from the amplified VH-encoding nucleic acid and theexact start and end point is identified by locating the amino acidsequences of EVQL/QVQL (SE ID NO: 40 and 41) to VSS (VH-region) (SEQ IDNO: 42) and DIVM/DIQM (SEQ ID NO: 43 and 44) to KLEIK (VL-region) (SEQID NO: 45).

Also reported herein is a method for producing an antibody comprisingthe following steps:

-   -   a) providing a population of (mature) B-cells (obtained from the        blood of an experimental non-human animal),    -   b) staining the cells of the population of B-cells with at least        one fluorescence dye (in one embodiment with one to four, or two        to four fluorescence dyes),    -   c) depositing single cells of the stained population of B-cells        in individual containers (in one embodiment is the container a        well of a multi well plate),    -   d) cultivating the deposited individual B-cells in the presence        of feeder cells and a feeder mix (in one embodiment the feeder        cells are EL-4 B5 cells, in one embodiment the feeder mix is        natural TSN, in one embodiment the feeder mix is a synthetic        feeder mix),    -   e) determining the binding specificity of the antibodies        secreted in the cultivation of the individual B-cells,    -   f) determining the amino acid sequence of the variable light and        heavy chain domain of specifically binding antibodies by a        reverse transcriptase PCR and nucleotide sequencing, and thereby        obtaining a monoclonal antibody variable light and heavy chain        domain encoding nucleic acid,    -   g) introducing the monoclonal antibody light and heavy chain        variable domain encoding nucleic acid in an expression cassette        for the expression of an antibody,    -   h) introducing the nucleic acid in a cell,    -   i) cultivating the cell and recovering the antibody from the        cell or the cell culture supernatant and thereby producing an        antibody.

In one embodiment the non-human animal is selected from rat, mouse,hamster, rabbit, non-human primates, sheep, dog, cow, chicken,amphibians, and reptiles.

The Method as Reported Herein

The invention is based at least in part on the finding that it isadvantageous to use EL4-B5 feeder cells in the co-cultivation withB-cells which have been obtained from a cultivation of EL4-B5 cells thathas a (final) cell density of more than 500,000 cells/ml, especially inthe range of 600,000 cells/ml up to 1,500,000 cells/ml.

One aspect as reported herein is a method for co-cultivating one or moreB-cells comprising the step of

-   -   incubating the one or more B-cells with EL4-B5 cells, whereby        the EL4-B5 cells have been obtained/are from a cultivation of        EL4-B5 cells that has a cell density of from 600,000 cells/ml up        to 1,500,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a cell density of650,000 cells/ml up to 1,450,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a cell density of650,000 cells/ml up to 825,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a cell density of700,000 cells/ml up to 775,000 cells/ml.

In one embodiment the cultivation of EL4-B5 cells has a cell density of1,400,000 cells/ml up to 1,500,000 cells/ml.

The method is exemplified in the following with two B-cell preparationsexpressing antibodies with different binding specificity.

In the first example B-cells obtained from a rabbit that had beenimmunized with human CDCP1 were used.

The B-cells obtained from the immunized rabbit were deposited as singlecells in the wells of a multi-well plate (for each three 96-well plates;252 wells per each EL4-B5 cell density). Each single deposited cell wasco-cultivated with 50,000 EL4-B5 cells irradiated with 50 gray in thepresence of TSN and SAC for 7 days.

The average frequency of IgG positive wells, i.e. the wells in which IgGcould be detected in the co-cultivation supernatant, was comparableirrespective of the cell density of the cultivation from which theEL4-B5 cells were taken (see following Table).

TABLE EL4-B5 cultivation cell 285 435 535 655 775 density [×1000/ml] IgGpositive wells per 49.6 42.4 39.7 38.9 42.1 plate [%]

Also the average productivity was comparable irrespective of the celldensity of the cultivation from which the EL4-B5 cells were taken (seefollowing Table).

TABLE EL4-B5 cultivation cell 285 435 535 655 775 density [×1000/ml]average IgG concentration 1.2 1.0 1.1 1.5 0.9 per plate [μg/ml]

The average frequency of antigen-specific IgG positive wells, i.e. thewells in which IgG was produced that specifically binds to the targetantigen, of the total wells was comparable only for low and high celldensities of the cultivation from which the EL4-B5 cells were taken (seefollowing Table).

TABLE EL4-B5 cultivation cell 285 435 535 655 775 density [×1000/ml]antigen-specific IgG wells 24.6 13.5 15.9 25.4 28.6 per plate [% totalwells]

The average frequency of antigen-specific IgG positive wells, i.e. thewells in which IgG was produced that specifically binds to the targetantigen, of the IgG positive wells was comparable only for the high celldensities of the cultivation from which the EL4-B5 cells were taken (seefollowing Table and FIG. 1).

TABLE EL4-B5 cultivation cell 285 435 535 655 775 density [×1000/ml]antigen-specific IgG wells 51.5 32.6 39.2 65.6 68.6 per plate [% IgG-positive wells]

In the second example B-cells obtained from a rabbit that had beenimmunized with human serum albumin were used.

The B-cells obtained from the immunized rabbit were deposited as singlecells in the wells of a multi-well plate (for each two 96-well plates;168 wells per each EL4-B5 cell density). Each single deposited cell wasco-cultivated with 50,000 EL4-B5 cells irradiated with 50 gray in thepresence of TSN and SAC for 7 days.

The average frequency of IgG positive wells, i.e. the wells in which IgGcould be detected in the co-cultivation supernatant, was comparable upto a cell density of 1,450,000 cells/ml in the cultivation from whichthe EL4-B5 cells were taken (see following Table).

TABLE EL4-B5 cultivation cell density [×1000/ml] 295 510 700 1100 14501710 2275 IgG positive wells per 70.3 61.3 65.5 67.3 53.0 38.1 55.4plate [%]

Also the average productivity was comparable up to a cell density of1,450,000 cells/ml in the cultivation from which the EL4-B5 cells weretaken (see following Table).

TABLE EL4-B5 cultivation cell density [×1000/ml] 295 510 700 1100 14501710 2275 average IgG 4.8 4.1 4.4 3.9 3.8 2.3 2.1 concentration perplate [μg/ml]

The average frequency of antigen-specific IgG positive wells, i.e. thewells in which IgG was produced that specifically binds to the targetantigen, of the total wells was comparable up to a cell density of1,450,000 cells/ml in the cultivation from which the EL4-B5 cells weretaken (see following Table).

TABLE EL4-B5 cultivation cell density [×1000/ml] 295 510 700 1100 14501710 2275 antigen-specific IgG 23.8 20.3 26.2 23.2 21.5 8.4 7.8 wellsper plate [% total wells]

The average frequency of antigen-specific IgG positive wells, i.e. thewells in which IgG was produced that specifically binds to the targetantigen, of the IgG positive wells was increased at cell densities ofabout 700,000 cells/ml and of about 1,450,000 cells/ml in thecultivation from which the EL4-B5 cells were taken (see following Tableand FIG. 2).

TABLE EL4-B5 cultivation cell density [×1000/ml] 295 510 700 1100 14501710 2275 antigen-specific IgG 35.0 33.2 40.0 34.5 40.2 21.6 14.7 wellsper plate [% IgG-positive wells]

The following examples and sequences are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

EXAMPLES

Materials and Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J., et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The molecularbiological reagents were used according to the manufacturer'sinstructions.

Media and Buffers

Blocking buffer for ELISA comprises 1×PBS and 1% BSA.

Coating buffer for ELISA comprises 4.29 g Na2CO3* 10 H2O and 2.93 gNaHCO3 add water to a final volume of 1 liter, pH 9.6 adjusted with 2 NHCl.

Ethanol-solution for RNA isolation comprises 70% Ethanol or 80% Ethanol.

FACS-buffer for immuno fluorescence staining comprises 1×PBS and 0.1%BSA.

IMDM-buffer for ELISA comprises 1×PBS, 5% IMDM and 0.5% BSA.

Incubation buffer 1 for ELISA comprises 1×PBS, 0.5 CroteinC.

Incubation buffer 2 for ELISA comprises 1X PBS, 0.5 CroteinC and 0.02%Tween 20.

Incubation buffer 3 for ELISA comprises 1×PBS, 0.1% BSA.

Incubation buffer 4 for ELISA comprises 1X PBS, 0.5% BSA, 0.05% Tween,PBS (10×), 0.01 M KH2PO4, 0.1 M Na2HPO4, 1.37 M NaCl, 0.027 M KCl, pH7.0.

PCR-buffer comprises 500 mM KCl, 15 mM MgCl2, 100 mM Tris/HCl, pH 9.0.

Wash buffer 1 for ELISA comprises 1×PBS, 0.05 Tween 20.

Wash buffer 2 for ELISA comprises 1×PBS, 0.1% Tween 20.

Wash buffer 3 for ELISA comprises water, 0.9% NaCl, 0.05 Tween 20.

EL-4 B5 medium comprises RPMI 1640 (Pan Biotech, Aidenbach, Germany)supplemented with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM Glutamine,1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mMsodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05mM β-mercaptoethanol (Gibco, Paisley, Scotland).

Animal Care and Immunization

The experimental animals were held according to the German animalprotection law (TierSCHG) as well as according to the respectiveEuropean guidelines.

The antigen was at first applied together with complete Freud's adjuvant(CFA). Further applications were with incomplete Freud's adjuvant (IFA).The antigen containing emulsion was applied subcutaneously whereby theemulsion comprised an amount of from 50 to 100 μg antigen depending onthe weight of the receiving experimental animal.

NZW rabbits (Charles River Laboratories International, Inc.) were usedfor immunization. The antigen was solved in K₃PO₄ buffer pH 7.0 at aconcentration of 1 mg/ml and mixed (1:1) with complete Freud's adjuvant(CFA) till generation of stabile emulsion. The rabbits received an intradermal (i.d.) injection of 2 ml of emulsion followed by a second intramuscular (i.m.) and third subcutaneous (s.c.)

injection each with 1 ml in one week interval. The fourth i.m. injectionof 1 ml was performed two weeks later followed by two further s.c.injections of 1 ml in four weeks interval.

During the immunization serum antibody titer was determined with anantigen specific assay. At an antibody titer with an IC₅₀ of 1:10000 theblood or the spleen of the immunized animal was removed. Forreactivation of antigen specific B-cells 30 μg to 50 μg of the antigenwas applied intravenously to the experimental animal three days prior tothe removal of the blood or the spleen.

Removal of Organs, Blood and Macrophages

Blood from mice and hamster was obtained by punctuation of theretrobulberic vein.

Blood from rabbits was obtained by punctuation of the ear vein or, forlarger volumes, of the ear artery. Whole blood (10 ml) was collectedfrom rabbits 4-6 days after the third, fourth, fifth and sixthimmunization and used for single cell sorting by FACS.

Macrophages were isolated from the obtained blood by attachment to cellculture plastic. From mice and hamsters, about 3*10⁵ macrophages can beobtained from each animal by this method.

If a larger amount of mouse or hamster macrophages was required,peritoneal macrophages were isolated. For this the animals have to be atleast 3 months of age. For the removal of peritoneal macrophages,animals were sacrificed and 5 ml of EL-4 B5 medium with a temperature of37° C. was immediately injected into the peritoneal cavity. Afterkneading the animal's belly for 5 minutes, the solution containing thecells was removed.

EDTA containing whole blood was diluted twofold with 1×PBS beforedensity centrifugation on lympholyte mammal (Cedarlane Laboratories) orFicoll Paque Plus (GE Healthcare, cat. #17-1440-03), which was performedto isolate rabbit PBMC. PBMCs were washed twice before staining withantibodies.

Density Gradient Centrifugation

The isolation of peripheral blood mononuclear cells (PBMCs) was effectedby density gradient separation with Lympholyte® according tomanufacturer's instructions A (Lympholyte®-mammal, Cedarlane).

Withdrawn blood was diluted 2:1 with phosphate buffered saline (PBS). Ina centrifuge vial the same volume of density separation medium wasprovided and the diluted blood is carefully added via the wall of thevial. The vial was centrifuged for 20 min. at 800×g without braking. Thelymphocytes were obtained from the white interim layer. The removedcells were supplemented with 10 ml PBS and centrifuged at 800×g for 10min. The supernatant was discarded and the pellet was resuspended,washed, centrifuged. The final pellet was resuspended in PBS.

Hypotonic Lysis of Red Blood Cells

For disruption of red blood cells by hypotonic lysis an ammoniumchloride solution (BD Lyse™) was diluted 1:10 with water and added at aratio of 1:16 to whole blood. For lysis of the red blood cells themixture was incubated for 15 min. in the dark. For separation of celldebris from intact cells the solution was centrifuged for 10 min. at800×g. The supernatant was discarded, the pellet was resuspended in PBS,washed again, centrifuged and the pellet was resuspended in PBS. Example8

Depletion of Macrophages

Sterile 6-well plates (cell culture grade) were used to depletemacrophages and monocytes through unspecific adhesion. Wells were eithercoated with KLH (keyhole limpet haemocyanine) or with streptavidin andthe control peptides. Each well was filled with 3 ml to (at maximum) 4ml medium and up to 6×10⁶ peripheral blood mononuclear cells from theimmunized rabbit and allowed to bind for 60 to 90 min. at 37° C. in theincubator. Thereafter the lymphocyte containing supernatant wastransferred to a centrifugation vial and centrifuged at 800×g for 10min. The pellet was resuspended in PBS.

Enrichment of antigen-specific B-cells

The respective antigen was diluted with coating buffer to a finalconcentration of 2 ng/ml. 3 ml of this solution were added to the wellof a 6-well multi well plate and incubated over night at roomtemperature. Prior to use the supernatant was removed and the wells werewashed twice with PBS. The B-cell solution was adjusted to a celldensity of 2×10⁶ cells/ml and 3 ml are added to each well (up to 6×10⁶cells per 3-4 ml medium) of a 6-well multi well plate. The plate wasincubated for 60 to 90 min. at 37° C. The supernatant was removed andnon-adherent cells were removed by carefully washing the wells 1-4 timeswith 1×PBS. For recovery of the sticky antigen-specific B-cells 1 ml ofa trypsin/EDTA-solution was added to the wells of the multi well plateand incubated for 10 to 15 min. at 37° C. The incubation was stopped byaddition of medium and the supernatant was transferred to acentrifugation vial. The wells were washed twice with PBS and thesupernatants were combined with the other supematants. The cells werepelleted by centrifugation for 10 min. at 800×g. The cells were kept onice until the immune fluorescence staining. The pellet was optionallyresuspended in PBS.

Co-cultivation of B-Cells and EL-4 B5 Cells

a) The co-cultivation was performed in 96-well multi well plates withround bottom. A basis solution comprising EL-4 B5 cells (1.6×10⁶cells/15.2 ml) and cytokines in EL-4 B5 medium was prepared. 200 μl ofthe basis solution was added to each well of the multi well plate. Toeach well a single B-cell was added by fluorescence activated cellsorting. After the addition of the B-cells the plate was centrifuged for5 min. at 300×g. The plate is incubated for seven days at 37° C.

b) Single sorted B-cells were cultured in 96-well plates with 210μl/well EL-4 B5 medium with Pansorbin Cells (1:20000) (Calbiochem(Merck), Darmstadt, Deutschland), 5% rabbit thymocyte supernatant andgamma-irradiated EL-4-B5 murine thymoma cells (2×10⁴/well) for 7 days at37° C. in an atmosphere of 5% CO₂ in the incubator. B-cell culturesupematants were removed for screening and the cells harvestedimmediately for variable region gene cloning or frozen at −80° C. in 100μl RLT buffer (Qiagen, Hilden, Germany).

Human B-cells were cultured accordingly.

Cultivation of T-Cells

The T-cells were isolated from the thymus of 3-4 week old mice andhamsters, or of 4-5 week old rabbits, respectively. The cells werecentrifuged and immediately cultivated or frozen in aliquots of 3×10⁷cells. The thymocytes were seeded with a minimum cell density of 5×10⁵cells/ml of EL-4 B5 medium in 175 cm² culture flasks and incubated for48 hours at 37° C.

Cultivation of Macrophages

Macrophages were isolated from the peritoneal cavity of mice andhamsters, respectively, of an age of at least three months. Peritonealmacrophages from mice or hamsters, or blood mononuclear cells fromrabbits were cultivated in EL-4 B5 medium at a cell density of at least1×10⁵ cells/ml in 175 cm² culture flasks for 1.5 hours at 37° C.Afterwards the medium was removed and non-attached cells were removedfrom the attached macrophages by washing with warm EL-4 B5 medium,followed by cultivation for 48 hours in 35 ml medium.

Co-Cultivation of T-Cells and Macrophages

T-cells and macrophages were cultivated for 48 hours in separate flasks.Prior to combining both cell populations, the T-cells were centrifugedfor 10 min. at 800×g. The supernatant was discarded and the cell pelletwas resuspended in 10 ml medium. The T-cells were adjusted to a minimalcell density of 5×10⁵ cells/ml and 10 pg phorbol-12-myristate-13-acetate(PMA) and 5 ng or 50 ng Phytohemagglutinin M (PHA-M) per ml of mediumwere added. The cultivation medium was removed from macrophages and theT-cell suspension was added to the flasks containing macrophages. After36 hours of co-cultivation, the cultivation medium was removed and wastermed TSN solution. For removal of remaining cells the TSN solution wasfiltered through a 0.22 μm filter. The TSN solution was frozen at −80°C. in aliquots of 4 ml.

Immunofluorescence Staining

Protocol 1:

Depending on the number of cells to be stained the cells were providedin 100 μl medium (less than 10⁶ cells) or 200 μl medium (more than 10⁶cells), respectively. The fluorescent labeled antibody was diluted with5% serum of the experimental animal and FACS buffer to a final volume of100 μl or 200 μl, respectively. The reaction mixture was incubated on aroller rack for 40 min. at 4° C. in the dark. After the incubation thecells were washed twice at 300×g for 5 min. The pellet was resuspendedin 400 μl PBS and filtered through a 70 μm sieve. The filtered solutionwas transferred to a FACS-vial and directly before the FACS experimentdead cells were stained by addition of propidium iodide (6.25 μg/ml). Ifthe labeled antibody was labeled with biotin the antibody was detectedin a second step with streptavidin labeled Alexa Flour(R) 647 (antibody197).

Protocol 2:

Anti-rabbit IgG FITC used for single cell sorting was from AbD Serotec(STAR121F, Dusseldorf, Germany).

For surface staining, cells were incubated with the optimally dilutedanti-rabbit IgG FITC antibody in PBS for 30 min. with rolling at 4° C.in the dark. Following centrifugation, the supernatants were removed byaspiration. The PBMCs were subjected to two cycles of centrifugation andwashing with ice cold PBS. Finally the PBMCs were resuspended in icecold PBS and immediately subjected to the FACS analyses. Propidiumiodide in a concentration of 5 μg/ml (BD Pharmingen, San Diego, Calif.,USA) was added prior to the FACS analyses to discriminate between deadand live cells. In other experiments the stained cells were singledeposited by FACS.

A Becton Dickinson FACSAria equipped with a computer and the FACSDivasoftware (BD Biosciences, USA) were used to collect and analyze thedata.

Proliferation Assays

a) Cell Titer Glo (CTG) viability assay

-   -   The CTG viability assay (Promega; #G7571) was used according to        the instructions of the manufacturer.

b) ³H Thymidine Assay

-   -   After 6 days of incubation ³H-Thymidin was added (0.5 μCi/well)        and incubated for further 16 hours. The incorporation of        ³H-Thymidine during cell proliferation was determined with a        microplate scintillation counter (Wallac).

c) Microscopic analysis

-   -   For the acquisition of microscopic images, a phase contrast        microscope from Leica (Leica DM IL) combined with a high        resolution camera (Leica DFC290 HD) was used.

d) Analysis of B-cell activation via CFSE-labeling.

-   -   Isolated B-cells were washed with sterile phosphate buffer        saline solution (PBS). Up to 1×10⁷ cells were resuspended in 1        ml protein-free PBS and incubated with CFSE (#C34554,        Invitrogen/Molecular Probes) for 3 to 10 minutes at a final        concentration of 2.5 μM at 37° C. CFSE loading was stopped by        addition of an excess of FCS-supplemented medium. After        extensive washing with FCS-containing medium, B-cells were used        in co-culture experiments. Proliferation of CD19⁺ gated        (B-)cells as a consequence of CFSE dilution was confirmed by        flow cytometric analysis (FL-1 channel) after indicated time        points.

Quantification of IgG

The 96-well multi well plate in which the co-cultivation was performedwas centrifuged after seven days of co-cultivation at 300×g for 5 min.150 μl supernatant was removed and diluted at a ratio of 2:1 with PBS ina second 96-well multi well plate.

The antibody was used at a concentration of 50 ng/ml. If the OD was orexceeded 1 after an incubation time of 5 min. a dilution series of from0.8 to 108 ng/ml IgG was tested.

Detection of Antigen-Specific IgG

Antibodies produced by single deposited and co-cultivated B-cells orfrom B-cells obtained from an immunized experimental animal can becharacterized with respect to specific antigen binding. The ELISA wasperformed at room temperature and the ELISA-solution was incubatedbetween the individual steps on a shaker at 20×g. In the first step theantigen was bound to the wells of a 96-well multi well plate. If theantigen was a protein it had been diluted in coating buffer and applieddirectly to the plate. Peptide antigens were bound via the specificbinding pair biotin/streptavidin. The wells of the multi well plate canbe already coated with soluble CroteinC (CrC) by the manufacturer. Ifnot, the wells were incubated after the immobilization of the antigenwith 200 μl blocking buffer. After the incubation with 100 μl antigensolution per well (pre-coated multi well plate) or 200 μl blockingbuffer, respectively, non-bound antigen or blocking buffer was removedby washing with wash buffer. The diluted B-cell supernatants were addedin a volume of 100 μl per well and incubated. After the incubation thewells were washed. Afterwards the detection antibody was added in avolume of 100 μl per well. The antibody can be either conjugated tohorseradish peroxidase or labeled with biotin. The detection antibodywas determined with a streptavidin-horseradish peroxidase conjugate.After the incubation the multi well plate was washed and afterwards 50μl of a substrate solution containing 3,3′,5,5′ tetramethyl benzidine(TMB) were added per well and incubated for a period as given in TableX. The enzymatic reaction was stopped by the addition of 50 μl sulfuricacid and the optical density was determined at 450 nm and 680 nm with aphotometer (Rainbow Thermo ELISA Reader) and the)(read plus-software.

Isolation of Ribonucleic Acid (RNA)

The cells from which the RNA had to be isolated were at first pelletedby centrifugation. The cell pellet was lysed by the addition of 100 μlRLT-buffer with 10 μl/ml beta-mercaptoethanol. The cells wereresuspended by multiple mixing with a pipette. The solution wastransferred to a well of a multi well plate. The plate was shortly shockat 200×g and frozen at −20° C.

The isolation of the RNA was performed with the RNeasy® Kit (Qiagen,Hilden, Germany) according to the manufacturer's instructions.

Reverse Transcription Polymerase Chain Reaction

Protocol 1:

The reverse transcription was carried out in a volume of 20 μl. For eachreaction a control was performed with and without reverse transcriptase.Per reaction 1 μl dNTP (each at 10 mM), 0.4 μl oligo(dT)₁₂₋₁₈ (0.2 μg)and 0.6 μl random hexamer (0.03 μg) were pre-mixed and added to 8.5 μlRNA in H2O. The reaction mixture was incubated for 5 min. at 65° C. anddirectly afterwards transferred to ice. Thereafter 2 μl RT-buffer (10×),4 μl MgCl2 (25 mM), 2 μl DTT (0.1 M) and 1 μl RNAse Out™ (40 units) werepre-mixed and added to the ice cold reaction mixture. After anincubation time of 2 min. at room temperature 0.5 μl Superscript™ IIreverse transcriptase (25 units) were added. The reaction mixture wasincubated for 10 min. at room temperature. The translation was carriedout for 50 min. at 42° C. After the translation the reversetranscriptase was inactivated by incubation for 15 min. at 70° C. ThecDNA was stored at −20° C.

Protocol 2:

cDNA was generated by reverse transcription of mRNA using the SuperScript III first-strand synthesis SuperMix (Invitrogen) according to themanufacturer's instructions. In a first step 6 μl of the isolated mRNAwas mixed with 1 μl annealing buffer and 1 μl (50 μM) oligo dT,incubated for 5 minutes at 65° C. and thereafter immediately placed onice for about 1 minute. Subsequently while still on ice 10 μl 2×First-Strand Reaction Mix and SuperScript™ III/RNaseOUT™ Enzyme Mix wereadded. After mixing the reaction was incubated for 50 minutes at 50° C.The reaction was terminated by incubation at 85° C. for 5 minutes. Aftertermination the reaction mix was placed on ice.

Polymerase Chain Reaction

Protocol 1:

The polymerase chain reaction was carried out with the Taq PCR Core Kit(Qiagen, Hilden, Germany) according to the manufacturer's instructions.The PCR was carried out in a volume of 20 μl. The samples weretransferred to the Mastercyler® at a temperature of 95° C.

Protocol 2:

The polymerase chain reaction was carried out using AccuPrime PfxSuperMix (Invitrogen) according to the manufacturer's instructions.Light chain and heavy chain variable regions were amplified in separatereactions. PCR-primer (0.2 μM/reaction) with 25 bp overlaps to targetantibody expression vectors were used. After the PCR 8 μl of the PCRreaction mixture were used for analysis on 48-well eGels (Invitrogen).

Sequencing

DNA sequences were determined by double strand sequencing performed atSequiServe GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

The GCG's (Genetics Computer Group, Madison, Wisconsin) software packagevariant 10.2 and Infomax's Vector NTI Advance suite variant 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Gene Synthesis

Desired gene segments encoding cDNA were prepared by Geneart GmbH(Regensburg, Germany). The gene segments are flanked by singularrestriction endonuclease cleavage sites to facilitate expressionconstruct cloning as described below. The DNA sequence of the subclonedgene fragments were confirmed by DNA sequencing.

Panning on Antigen

a) Coating of plates

Biotin/Streptavidin: Sterile streptavidin-coated 6-well plates (cellculture grade) were incubated with biotinylated antigen at aconcentration of 0.5-1(2) μg/ml in PBS at room temperature for one hour.Plates were washed in sterile PBS three times before use.

Covalently bound protein: Sterile cell culture 6-well plates were coatedwith 2 μg/ml protein in carbonate buffer (0.1 M sodium bicarbonate, 34mM disodium hydrogen carbonate, pH 9.55) over night at 4° C. Plates werewashed in sterile PBS three times before use.

b) Panning of B-cells on antigen

6-well tissue culture plates coated with the respective antigen wereseeded with up to 6×10⁶ cells per 4 ml medium and allowed to bind forone hour at 37° C. in the incubator. Non-adherent cells were removed bycarefully washing the wells 1-2 times with 1×PBS. The remaining stickycells were detached by trypsin for 10 min.

at 37° C. in the incubator and then washed twice in media. The cellswere kept on ice until the immune fluorescence staining.

Preparation of Plasmid-DNA

The plasmid DNA to be used as recipient for the cloning of the PCRproducts encoding the antibody heavy and light chain variable domainswas first linearized by restriction enzyme digestion. Subsequently, thelinearized plasmid DNA was purified by preparative agaroseelectrophoresis and extracted from the gel (QIAquick Gel ExtractionKit/Qiagen). This purified plasmid DNA was added to a PCR-protocol astemplate using primer overlapping (by 20-25 bp) with the PCR-products tobe cloned. The PCR was carried out using AccuPrime Pfx SuperMix(Invitrogen).

Cloning

The PCR-products were cloned into expression vectors using a “site andligation independent cloning” method (SLIC) which was described by Haun,R. S., et al. (BioTechniques 13 (1992) 515-518) and Li, M. Z., et al.(Nature Methods 4 (2007) 251-256). Both purified vector and insert weretreated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim,Germany) per 1 μg DNA for 45 minutes at 25° C. in the absence of dNTPsto generate matching overhangs. The reaction was stopped by adding1/10^(th) of the reaction volume of a 10 mM dCTP Solution (Invitrogen).The T4 treated vector and insert DNA fragments were combined with aplasmid:insert ratio of 1:2 (w/w) (e.g. 100 ng:200 ng) and recombined byadding RecAProtein (New England Biolabs) and 10× RecA Buffer for 30minutes at 37° C. Subsequently, 5 μl of each of the generated heavychain and light chain expression plasmid was used to transform MultiShotStrip Well TOP 10 Chemically Competent E. coli cells (Invitrogen) usinga standard chemical transformation protocol. After regeneration (shakingfor 45 minutes at 37° C. of the transformed E. coli cells) the entiretransformation mixture was transferred into DWP 96 (deep well plates)containing 2 ml of LB medium supplemented with ampicillin per well. Thecells were incubated in a shaker for 20 hours at 37° C. In the followingstep the plasmid DNA encoding the immunoglobulin heavy- and light chainswas purified using the NucleoSpin 96 Plasmid Mini Kit (Macherey&Nagel),digested with selected restriction enzymes, and analyzed on 48-welleGels (Invitrogen). In parallel, glycerol stocks were prepared forstorage.

Transfection and Expression of Recombinant Antibodies in EukaryoticCells

HEK293 cells were grown with shaking at 120 rpm in F17-medium (Gibco) at37° C. in an atmosphere containing 8% CO₂. Cells were split the daybefore transfection and seeded at a density of 0.7-0.8×10⁶ cells/ml. Onthe day of transfection, 1-1.5×10⁶ HEK293 cells in a volume of 2 ml weretransfected with 0.5 μg HC plasmid plus 0.5 μg LC plasmid, suspended in1 μl 293-free medium (Novagen) and 80 μl OptiMEM® medium (Gibco) in 48well deep well plates. Cultures were incubated for 7 days at 180 rpm at37° C. and 8% CO₂. After 7 days the culture supernatants were harvested,filtered and analyzed for antibody content and specificity.

Primer

a) Primer for B-cell PCR of B-cells expressing rabbit antibodies

primer set 1: LC-primer rb-V-kappa-HindIIIs (SEQ ID NO: 01):GATTAAGCTTATGGACAYGAGGGCCCCCACTC rb-C-kappa-NheIas (SEQ ID NO: 02):GATCGCTAGCCCTGGCAGGCGTCTCRCTCTAACAG HC-primerrb-CH1rev-1 (SEQ ID NO: 03): GCAGGGGGCCAGTGGGAAGACTGrbVH3-23for3 (SEQ ID NO: 04): CACCATGGAGACTGGGCTGCGCTGGCTTCprimer set 2: LC-primer rb-V-kappa-Slic-s001 (SEQ ID NO: 06):AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC rbCk1-rev2 (SEQ ID NO: 07):CAGAGTRCTGCTGAGGTTGTAGGTAC HC-primerrb-VH3-23-Slic-s001 (SEQ ID NO: 08):AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC rb-CH1rev-2 (SEQ ID NO: 09):CCATTGGTGAGGGTGCCCGAG

-   -   primer for amplification of heavy chain expression plasmid        backbone:

8001-Slic-s001 (SEQ ID NO: 10): TGGGAACTCGGGCACCCTCACCAATGG8001-Slic-as002 (SEQ ID NO: 11):GCCCAGTCTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCTG

-   -   primer for amplification of kappa light chain expression plasmid        backbone:

8011-Slic-s001 (SEQ ID NO: 12): GTACCTACAACCTCAGCAGCACTCTG8000-Slic-as002 (SEQ ID NO: 13):CCCTCRTGTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCTG

b) Primer for B-cell PCR of rabbit B-cells expressing human antibodies(derived from transgenic rabbit)

-   -   primer for amplification of heavy chain variable domains

HC-Up rb-VH3-23-Slic-s001 (SEQ ID NO: 08):AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTCbcPCR-huCgamma-rev (SEQ ID NO: 05): CCCCCAGAGGTGCTCTTGGA

-   -   primer for amplification of light chain variable domains

bcPCR-FHLC-leader-fw (SEQ ID NO: 06): ATGGACATGAGGGTCCCCGCbcPCR-huCkappa-rev (SEQ ID NO: 07): GATTTCAACTGCTCATCAGATGGC

c) Primer for the amplification of heavy chain plasmid backbone:

bcPCR-hu-HC-10600-SLIC-as (SEQ ID NO: 08):CAGCCCAGTCTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCTGbcPCR-hu-HC-10600-SLIC-s (SEQ ID NO: 09): CTCCAAGAGCACCTCTGGGGGCACAG

d) Primer for the amplification of kappa light chain plasmid backbone:

bcPCR-hu-LC-10603-SLIC-s (SEQ ID NO: 10): GCCATCTGATGAGCAGTTGAAATCbcPCR-hu-LC-10603-SLIC-as (SEQ ID NO: 11):GCGGGGACCCTCATGTCCATGGTGGCAAGCTTCCTCTG

e) Primer for B-cell PCR of B-cells from human donors

-   -   primer for amplification of heavy chain variable domains

SLIC-hu-VHuniversal-for (SEQ ID NO: 12):AGCAACAGCTACAGGTGTGCATTCCGAGGTGCAGCTGKTGSAG TCTGSSLIC-hu-VH6-for (SEQ ID NO: 13):AGCAACAGCTACAGGTGTGCATTCCCAGGTRCAGCTGCAGSAG TChu-CH1gamma-rev (SEQ ID NO: 14): GTCCACCTTGGTGTTGCTGGGCTT

-   -   Primer for amplification of kappa light chain variable domains

SLIC-huVk2-for (SEQ ID NO: 15):TAGCAACAGCTACAGGTGTGCATTCCGATGTTGTGATGACTCAG TCTSLIC-huVk3-for (SEQ ID NO: 16):TAGCAACAGCTACAGGTGTGCATTCCGAAATTGTGWTGACRCA GTCTSLIC-huVk5-for (SEQ ID NO: 17):TAGCAACAGCTACAGGTGTGCATTCCGACATCGTGATGACCCA GSLIC-huVk7-for (SEQ ID NO: 18):TAGCAACAGCTACAGGTGTGCATTCCGAAATTGTGCTGACTCA GTCTSLIC-huVk8-for (SEQ ID NO: 19):TAGCAACAGCTACAGGTGTGCATTCCGAWRTTGTGMTGACKCA GTCTCCSLIC-huVk1long-for (SEQ ID NO: 20):TAGCAACAGCTACAGGTGTGCATTCCGACATCCRGWTGACCCA GTCTSLIC-huVk2longw-for (SEQ ID NO: 21):TAGCAACAGCTACAGGTGTGCATTCCGATRTTGTGATGACYCA GWCThuCk-rev (SEQ ID NO: 22): ACACTCTCCCCTGTTGAAGCTC

-   -   Primer for amplification of lambda light chain variable domains

SLIC-huVl1-for (SEQ ID NO: 23):TAGCAACAGCTACAGGTGTGCATTCCCAGTCTGTGYTGACKCAGSLIC-huVl2-for (SEQ ID NO: 24):TAGCAACAGCTACAGGTGTGCATTCCCAGTCTGCCCTGACTCAGSLIC-huVl3-for (SEQ ID NO: 25):TAGCAACAGCTACAGGTGTGCATTCCTCCTATGAGCTGAYWCAGSLIC-huVl4-for (SEQ ID NO: 26):TAGCAACAGCTACAGGTGTGCATTCCCAGCYTGTGCTGACTCAASLIC-huVl5-for (SEQ ID NO: 27):TAGCAACAGCTACAGGTGTGCATTCCCAGSCTGTGCTGACTCAGSLIC-huVl6-for (SEQ ID NO: 28):TAGCAACAGCTACAGGTGTGCATTCCAATTTTATGCTGACTCAGSLIC-huVl7-for (SEQ ID NO: 29):TAGCAACAGCTACAGGTGTGCATTCCCAGRCTGTGGTGACTCAGSLIC-huVl8-for (SEQ ID NO: 30):TAGCAACAGCTACAGGTGTGCATTCCCAGACTGTGGTGACCCAGSLIC-huVl9-for (SEQ ID NO: 31):TAGCAACAGCTACAGGTGTGCATTCCCWGCCTGTGCTGACTCAGSLIC-huVlambda10-for (SEQ ID NO: 32):TAGCAACAGCTACAGGTGTGCATTCCCAGGCAGGGCTGACTCAG huCl-1-rev (SEQ ID NO: 33):TCTCCACGGTGCTCCCTTC

f) Primer for amplification of human immunoglobulin expression plasmid

-   -   amplification of heavy chain expression plasmid backbone:

huIg-PCR-vectorprimer-as (SEQ ID NO: 34): GGAATGCACACCTGTAGCTGTTGCTAhuIg-PCR-vectorprimer-VH-s (SEQ ID NO: 35): AAGCCCAGCAACACCAAGGTGGAC

-   -   amplification of kappa light chain expression plasmid backbone:

huIg-PCR-vectorprimer-as kappa (SEQ ID NO: 36):GGAATGCACACCTGTAGCTGTTGCTA huIg-PCR-vectorprimer-VK-s (SEQ ID NO: 37):GAGCTTCAACAGGGGAGAGTGT

-   -   amplification of lambda light chain expression plasmid backbone:

huIg-PCR-vectorprimer-as lambda (SEQ ID NO: 38):GGAATGCACACCTGTAGCTGTTGCTA huIg-PCR-vectorprimer-VL-s (SEQ ID NO: 39):GAAGGGAGCACCGTGGAGA

Cytokines

Zubler Mix: 2 ng/ml mouse IL-1β, 50 ng/ml mouse IL-2, 10 ng/ml mouseIL-10, and 2 ng/ml mouse TNFα (final concentration)

Cytokines tested for establishment of a defined cytokine cocktail (givenas final concentration in case not stated otherwise):

final cytokine concentration supplier Catnr. huIL-2  50 U/ml Roche Dia.GmbH 11147528001 huIL-21   25 ng/ml eBioscience 14-8219 muIL-21  100ng/ml R&Dsystems 594-ML huIL-6 300 U/ml Roche Dia. GmbH 11138600001huIL-10   25 ng/ml BD 554611 huIL-1β 12.5 ng/ml R&Dsystems 201-LBhulL-33  100 ng/ml Peprotech 200-33 TNFα   25 ng/ml R&Dsystems 210-TA

Rabbit B-Cell Medium

500 ml RPMI 1640 #P04-17500 PAN Biotech  50 ml FCS #A15-512 PAA  5 mlL-Gln #25030-024 Invitrogen  5 ml potassium Pyruvate #P04-43100 PANBiotech  5 ml HEPES #15630-056 Invitrogen 500 μl β-Mercaptoethanol#31350010 Invitrogen  1 ml Pen/Strep #11074440001 Roche Dia. GmbH

Additives to Rabbit B-Cell Medium

SAC #507858 Calbiochem IL-21 #14-8219 eBioscience IL-2 #1114752800 Roche96er U-plate #3799 Coming

Phenotyping/Sorting of Antibodies

goat anti-rabbit IgG Fc-antibody AbDSerotec STAR121F rat anti-rabbitCD138-antibody Roche GlycArt AG anti-human CD40 mAb Beckman CoulterIM1374 (clone Mab89) anti human/murine (rabbit cross-reactive) antiCD40L antibody: anti-muCD40L antibody R&D systems AF1163 anti-huCD40Lantibody &D systems AF617 R donkey anti-goat IgG antibody MolecularProbes A11055 Alexa 488

Miscellaneous

anti-FITC antibody-coupled Miltenyi #130-048-701 microbeads Biotec humanB-cell negative Invitrogen #113.13D isolation kit Nucleofector Kit TLonza VCA-1002 CBA for total IgG BD #558679 Biosciences

Example 1

Cultivation of EL-4 B5 Cells

The frozen EL-4 B5 cells were thawed rapidly in a water bath at 37° C.and diluted with 10 ml EL-4 B5 medium. After centrifugation at 300×g for10 minutes the supernatant was discarded and the pellet resuspended in 1ml medium.

The EL-4 B5 cells were inoculated at a cell density of 8×10 cells/ml inT175 cultivation flasks. Cell density was determined every second dayand adjusted to 8×10⁴ cells/ml. The cells have a doubling time ofapproximately 18 hours. Afterwards cells were seeded with cell countsfrom 3×10⁴ to 4×10⁵ cells/ml to reach different final cell densities ofup to 2275×10⁵ cells/ml.

Cells were harvested and adjusted to a cell density of 1×10⁶ cells/mlbefore γ-irradiation at 50 Gy.

Example 2

Co-Cultivation of B-Cells and EL-4 BS Cells

Single sorted B-cells were cultured in 96-well plates with 200 μl/wellEL-4 B5 medium with Pansorbin Cells (1:100000) (Calbiochem (Merck),Darmstadt, Deutschland), 5% rabbit thymocyte supernatant andgamma-irradiated EL-4-B5 murine thymoma cells (5×10⁴/well) for 7 days at37° C. in an atmosphere of 5% CO₂ in an incubator. B-cell culturesupernatants were removed for screening and the cells harvestedimmediately for variable region gene cloning or frozen at −80° C. in 100μl RLT buffer (Qiagen, Hilden, Germany).

1. A method for co-cultivating one or more B-cells comprising the stepsof cultivating EL4-B5 cells to a cell density of from 600,000 cells/mlto 1,500,000 cells/ml, and incubating the one or more B-cells with analiquot of the EL4-B5 cells obtained in the previous step.
 2. The methodaccording to claim 1, wherein the cultivation of the EL4-B5 cells is toa cell density of 650,000 cells /ml to 1,450,000 cells/ml.
 3. The methodaccording to claim 1, wherein the cultivation of the EL4-B5 cells is toa cell density of more than 1,000,000 cells/ml to 1,500,000 cells/ml. 4.The method according to claim 1, wherein the cultivation of the EL4-B5is to a cell density of 650,000 cells/ml to 825,000 cells/ml.
 5. Themethod according to claim 1, wherein the cultivation of the EL4-B5 cellsis to a cell density of 1,400,000 cells/ml to 1,500,000 cells/ml.
 6. Themethod according to claim 1, wherein the incubating is additionally inthe presence of a feeder mix.
 7. The method according to claim 6,wherein the feeder mix comprises one or more of: i) interleukin-1 betaand tumor necrosis factor alpha, ii) interleukin-2 (IL-2) and/orinterleukin-10 (IL-10), iii) Staphylococcus aureus strain Cowan's cells(SAC), iv) interleukin-21 (IL-21) and optionally interleukin-2 (IL-2),v) B-cell activation factor of the tumor necrosis factor family (BAFF),vi) interleukin-6 (IL-6), vii) interleukin-4 (IL-4), and viii) thymocytecultivation supernatant.
 8. The method according to claim 6, wherein thefeeder mix comprises IL-1β, TNF-α, IL-10 and one or more of the membersselected from the group consisting of: IL-21, SAC, BAFF, IL-2, IL-4, andIL-6.
 9. The method according to claim 1, wherein the EL4-B5 cells are,after the cultivating and prior to the incubating, irradiated withγ-radiation.
 10. The method according to claim 1, wherein the method isfor the co-cultivation of one B-cell.
 11. The method according to claim10, wherein the one B-cell is a single deposited B-cell.
 12. The methodaccording to claim 1, wherein the incubating is for 5 to 14 days.
 13. Amethod for producing an antibody, the method comprising performing themethod according to claim 1, thereby producing the antibody.
 14. Themethod according to claim 13, wherein the antibody is produced by theone or more B cells.
 15. The method according to claim 14, wherein theantibody is produced by the one or more B cells and secreted into cellculture supernatant.
 16. The method according to claim 14, furthercomprising recovering the antibody from the one or more B cells.
 17. Themethod according to claim 15, further comprising recovering the antibodyfrom the cell culture supernatant.